Moraxella (Branhamella) catarrhalis bacteria are Gram-negative diplococcal pathogens which are carried asymptomatically in the healthy human respiratory tract. In recent years, M. catarrhalis has been recognized as an important causative agent of otitis media. In addition, M. catarrhalis has been associated with sinusitis, conjunctivitis, and urogenital infections, as well as with a number of inflammatory diseases of the lower respiratory tract in children and adults, including pnuemonia, chronic bronchitis, tracheitis, and emphysema (refs. 1 to 8). (Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Occasionally, M. catarrhalis invades to cause septicaemia, arthritis, endocarditis, and meningitis (refs 9 to 13).
Otitis media is one of the most common illnesses of early childhood and approximately 80% of all children suffer at least one middle ear infection before the age of three (ref. 14). Chronic otitis media has been associated with auditory and speech impairment in children, and in some cases, has been associated with learning disabilities. Conventional treatments for otitis media include antibiotic administration and surgical procedures, including tonsillectomies, adenoidectomies, and tympanocentesis. In the United States, treament costs for otitis media are estimated to be between one and two billion dollars per year.
In otitis media cases, M. catarrhalis commonly is co-isolated from middle ear fluid along with Streptococcus pneumoniae and non-typable Haemophilus influenzae, which are believed to be responsible for 50% and 30% of otitis media infections, respectively. M. catarrhalis is believed to be responsible for approximately 20% of otitis media infections (ref. 15). Epidemiological reports indicate that the number of cases of otitis media attributable to M. catarrhalis is increasing, along with the number of antibiotic-resistant isolates of M. catarrhalis. Thus, prior to 1970, no β-lactamase-producing M. catarrhalis isolates had been reports, but since the mid-seventies, and incresing number of β-lactamase-expressing isolates have been detected. Recent surveys suggest that 75% of clinical isolates produce β-lactamase (ref. 16, 26).
Iron is an essential nutrient for the growth of many bacteria. Several bacterial species, including M. catarrhalis, obtain iron from the host by using transferring receptor proteins to captures transferrin. A number of bacteria including Neisseria meningitidis (ref. 17), N. gonorrhoeae (ref. 18), Haemophilus influenzae (ref. 19), as well as M. catarrhalis (ref. 20), produce outer membrane proteins which specifically bind human transferrin. The expression of these proteins is regulated by the amount of iron in the environment.
The two transferrin receptor proteins of M. catarrhalis, designated transferrin binding protein 1 (Tbp1) and transferrin binding protein 2 (Tbp2), have molecular weights of 115 kDa (Tbp1) and approximately 80 to 90 kDa (Tbp2). Unlike the transferrin receptor protein of other bacteria which have an affinity for apotransferrin, the M. catarrhalis Tbp2 receptors have a preferred affinity for iron-saturated (i.e., ferri-) transferrin (ref. 21).
M. catarrhalis infection may lead to serious disease. It would be advantageous to provide a recombinant source of transferrin binding proteins as antigens in immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents. The genes encoding transferrin binding proteins and fragments thereof are particularly desirable and useful in the specific identification and diagnosis of Moraxella and for immunication against disease caused by M. catarrhalis and for the generation of diagnositc reagents.
There had previously been described in published PCT appliation WO 97/32380, assigned to Connaught Laboratories Limited, the assignee hereof, the cloning, subcloning and sequencing of nucleic acid molecules encoding transferrin receptor proteins Tbp1 and Tbp2 of certain specific strains of Moraxella catarrhalis, namely M. catarrhlais strains 4223, Q8 and R1, as well as identifying the deduced amino acid sequences of the encoded Tbp1 and Tbp2 proteins.
WO 97/32380 further describes the construction of expression plasmids for the production of recombinant Tbp1 from M. catarrhalis strain 4223 and of recombinant Tbp2 from M. catarrhalis strains 4223 and Q8, the recombinant expression of such proteins in E. coli, and the extraction and purification of the expressed Tbp1 and Tbp2 proteins.